Parietaria judaica ns-ltp antigen variants, uses thereof and compositions comprising them

ABSTRACT

The present invention relates to hypoallergenic variants of ns-LTPs allergens, to pharmaceutical compositions comprising them and to the use of such variants for the preparation of medicaments suitable in the treatment and in the prevention of the allergic forms associated with an ns-LTP allergen, in particular to the allergen corresponding to the variant used.

FIELD OF THE INVENTION

The present invention relates to the fields of the prevention and thetreatment of allergic symptoms associated with allergens belonging tothe non-specific Lipid Transfer Protein (ns-LTPs) family.

STATE OF THE ART

Ns-LTPs proteins are small proteic molecules of approximately 10 KDathat demonstrate high stability, and are naturally present in allvegetal organisms studied to date. In several species they have alsobeen identified as allergens, as in the case of the Rosaceae Prunoideae(peach, apricot, plum) and Pomoideae (apple), and Graminaceae, as in theUrticaceae like Parietaria Judaica (18-23).

These proteins are characterized by their ability to transport lipidsthrough membranes in vitro, an ability justifying their denomination andcorresponding to at least some of the activities exerted in vivo (17).

However, in spite of the different functions and of the heterogeneity oftheir sequence, ns-LTPs have a highly conserved secondary structure,comprising four alpha-helices (separated by loops) and one folded betalayer arranged in the 5′-3′ direction according to a α-α-α-α-β pattern.

This structure is provided by the presence of four disulfide bridgesformed by eight cysteine residues present in the 4 alpha-helices in thefourth loop, in the folded beta-layer and in the amino-terminal region(cfr. ref. 29).

In particular:

-   -   a first disulfide bridge connects the amino-terminal region and        the third alpha-helix,    -   a second disulfide bridge connects the first alpha-helix and the        third alpha-helix,    -   a third disulfide bridge connects the second alpha-helix and the        fourth loop, and    -   a fourth disulfide bridge connects the third alpha-helix and the        folded beta-layer.

These cysteine residues are highly conserved in all ns-LTPs, and withreference thereto a consensus sequence can be derived (17).

Despite their high conservation, given the sequence heterogeneity ofns-LTPs, no notation system for the residues forming disulfide bridgesvalid for all ns-LTPs exists, though those skilled in the art may easilysingle out such cysteines using the knowledge of the state of the art.

With particular reference to the Parj1 protein, and specifically to themature ParJ1.0102 form, the cysteines apt to form disulfide bridges arethe residues 4, 14, 29, 30, 50, 52, 75 and 91, and the related bridgesare arranged in the order Cys4-Cys52 (first bridge), Cysl4-Cys29 (secondbridge), Cys30-Cys75 (third bridge), Cys50-Cys91 (fourth bridge) (12).

Par J1, besides being an ns-LTP, represents, together with Par J2, oneof the major allergens of the Parietaria Judaica (PJ), a plant whosepollen constitutes one of the most widespread environmental antigens,especially in the Mediterranean area (1).

In fact, Parietaria Judaica pollen contains at least nine allergens withmolecular masses ranging from 10 to 80 KDa and different capabilities ofbinding IgE [1-9].

Thereamong, Parj1, in the two isoform parl1.1.2 and Parj1.0201 isolatedfrom independent genes (10) and Parj2 acquire a remarkable relevance asmajor allergens. In particular these two ns-LTPs are capable ofinhibiting the majority of specific IgE against Parietaria allergens,and, upon administration, both have an immunological behaviour in allalike that of the commercial extracts commonly used (11).

However, ParJ1 and ParJ2 do not constitute the sole ns-LTPs havingallergenic properties. Recently, some scientific papers describing thecharacterisation of new allergenic molecules homologous to the ns-LTPshave been published (18-23).

Despite sequence heterogeneity, following cross-reactivity experimentsbetween different ns-LTP allergens and related produced antibodies, itwas demonstrated that ns-LTP constitute a widespread family of allergens(pan-allergen) as already described for profilin (19).

However, in comparison with the abundant information on the structure ofthis ‘pan-allergen’, exhaustive information on the localisation of theepitopes for IgE and IgG therein, as well as in the individual ns-LTPallergens (Parj1 and ParJ2 included) are not available (11, 12).

As a result of the mechanism in charge of the development of theallergic response, and of the verified role of IgG and IgE therein, thederivation of such a map would be instead of enormous relevance for thedrafting of a novel therapeutic approach to these allergic forms (13).

In particular, the derivation of molecules with reduced or even absentIgE binding capability, yet concomitantly capable of inducing IgGresponse, and in particular of IgG4, might be a landmark both from atherapeutic and a preventive point of view.

Such a molecule would allow immunosuppression of the T cell responsewith reduced or even absent side effects.

In fact, to date the therapy of an undergoing allergy consists in themere pharmacological cure of the allergic symptomatology.

A preventive therapy represented by the specific immunotherapy (SIT)actually consists in the subcutaneous administration of dilutedquantities of allergen to the patient so as to suppress the specificreaction towards the allergen.

The majority of the commercial protein extracts used therefor however,are anyhow crude extracts, mixtures of several components in which aprecise standardization of the allergenic component is difficult.

Thus, the SIT strategy can entail the administration of allergeniccomponents towards which the patient is not sensitive, inducing thesecretion of IgEs specific towards other components of the extract.Moreover, the administration of the total allergen entails thepossibility of side effects which, though with extremely low occurrence,could even cause anaphylactic shock.

Concerning in particular the Parietaria Judaica, epidemiological studieshave also highlighted a different distribution of the two majorallergens in the human population (12 millions of affected subjects inthe Mediterranean area) where, approximately 20% of the PJ allergicpatients do not exhibit a concomitant presence of IgE specific againstboth allergens. Therefore, an administration of total or partiallypurified crude extracts could entail an administration of majorallergens to which the patient is not allergic.

Hence, the use of recombinant molecules, allowing a patient customizeddiagnosis and therapy, could represent a valid alternative to thetraditional use of crude extracts.

In particular, the characterization and the development of alternativemolecules with reduced side effects, i.e., having a reduced or absentinteraction with the IgE while maintaining the capability of binding theIgGs (in particular the IgG4) with respect to the wild type andtherefore the capability of immunosuppressing the T response, couldallow to implement an alternative approach overcoming the disadvantagesinherent to the traditional approach.

Such an alternative molecule with reduced anaphylactic capacity were infact sought by producing crude formaldehyde- orglutaraldehyde-polymerised extracts (16).

Although effective, as demonstrated by clinical trials, these modifiedmolecules have proved however to present the abovedescribed disadvantageof a difficult standardization of the extracts.

Following the advent of genetic engineering both recombinant allergensimmunologically similar to the native allergens (14 and 15), andrecombinant allergen having instead a reduced allergenic activity withrespect to the allergen wild type (therefore therapeutically suitable asa substitute of the latter), have been derived in a pure form.

None of such a mutant have not however been derived with particularreference to the ns-LTPs allergens.

SUMMARY OF THE INVENTION

An object of the present invention is a variant of an allergen belongingto the ns-LTP protein family, specifically a hypoallergenic variant of acomplete allergen or fragment thereof of the ns-LTD protein family.

In particular object of the invention is a variant of an allergenbelonging to the ns-LTP family which lacks at least one of the fourdisulfide bridges constituting the structure of said allergen.

A first advantage of the variant of the present invention is that withrespect to the native allergen it has a reduced or even absentcapability of binding IgE, having concomitantly an intact capability ofbinding IgG of the said subjects.

This differential binding capability is particularly enhanced in thevariants wherein such a missing bridge be localized in theamino-terminal region of the allergen at the domain alpha-helix 1-loop1-alpha-helix 2, as they have a particularly reduced IgE bindingactivity, especially in the variants lacking at least two disulfidebridges.

In case such a variant be lacking three, or all four, of the disulfidebridges of the native allergen, the relevant IgE binding activity isreduced up to be substantially absent. Such a variant constitutesaccordingly a preferred embodiment of the invention.

The relevance of such a differential binding capability of the variantof the invention lies in that according to the role of the twoimmunoglobulins in the molecular mechanism of the allergic responseevidenced in the above paragraph, it turns out in molecules, thatalthough immunogenic have a reduced or absent allergenicity.

Variants of the invention which mainly maintain most of amino acidsequence of the wild type allergen, and has accordingly substiantiallythe same length of the said allergen, constitute in this connection apreferred embodiment of the invention.

In particular variants consisting of a mutein in which at least one ofthe cysteines constituting said disulfide bridges is deleted, orsubstituted with an amino acid residue not capable of forming disulfidebridges, are preferred.

In this latter case, the substitution of the cysteine residue withserine or alanine, as amino acids tested compatible with the ns-LTPα-α-α-α-5 structure, proved particularly effective.

The embodiment related to variants of the major allergens of ParietariaJudaica is particularly preferred.

In particular, the Parj1 variants, specifically the Parj1 muteins inwhich the deleted or substituted residues are the cysteines 4, 14, 29,30, 50, 52, 75 and 91, and in particular the variants having a sequenceselected in the group comprising the sequences reported in the sequencelisting as SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10,are especially relevant.

Object of the present invention is also a polynucleotide coding for thevariants of the present invention, in particular for the above indicatedmuteins, and specifically the polynucleotides comprising a sequenceselected in the group comprising the sequences reported in the sequencelisting as SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, aswell as the vectors comprising them.

In the light of what set forth above, object of the present inventionare also any of the above mentioned variants for use as medicament or asa diagnostic agent, and in particular for use in the treatment and/orthe prevention and/or the diagnosis of the allergic form associated withan allergen belonging to the family of ns-LTP proteins, (this in lightof the pan-allergen characteristic verified for the ns-LTP allergens),in particular to the allergen corresponding to said variant.

In the specific case, hereto disclosed by way of example, of majorallergens of Parietaria Judaica showing an uneven ability to stimulateserum IgE production in allergic patients, a specific diagnosis for theindividual allergen may be attained using the variants of the invention,e.g., as follows: initially the recombinant version of each nativemolecule (Parj1 and Parj2) is used for a specific diagnosis of theallergy by skin prick test. Patients sensitive to one of two allergenscan then be analyzed for positiveness to the individual allergenvariants to which they tested positive in order to highlight negativelytesting variants. Then, such variants can be administered insubstitution of commercial protein extracts developing anallergen-specific immunotherapy. Further modes of employ for diagnosticas well as therapeutic ends are anyhow derivable by those skilled in theart in light of the knowledge of the state of the art.

Object of the present invention is also a pharmaceutical compositioncomprising a therapeutically effective quantity of at least one of thevariants, or a polynucleotide or a vector among the above mentionedones, and a pharmaceutically acceptable carrier, as well as all thematter compositions comprising at least one of the above mentionedmolecules and one carrier chemically compatible therewith.

This pharmaceutically and/or chemically acceptable carrier can be anyone carrier known to the art as suitable in pharmaceutical or mattercompositions containing the molecules like the above mentioned ones,therefore in particular peptides and conjugates and/or oligonucleotidesin any form, in particular in solid and in liquid form; an example ofcomposition in liquid form is provided by compositions whose carrier iswater, saline solutions, like, e.g., solutions containing NaCl and/orfosfate, or other solutions containing buffer molecules.

A still further object of the present invention is a kit for thederivation of a subject-customised allergogram, for an allergic formassociated with an ns-LTP allergen comprising

-   -   a first composition comprising said ns-LTP allergen in native        form together with a chemically and/or pharmaceutically        acceptable carrier;    -   at least one composition comprising a single variant of said        ns-LTP allergen as abovedescribed and a chemically and/or        pharmaceutically acceptable carrier;    -   said allergogram being derivable contacting said compositions        with immunoglobulins of said subject and observing the effects        thus obtained.

Particularly preferred are the embodiments in which the kit comprises,besides said first composition, a number of compositions each comprisinga single variant of said ns-LTP allergen, equal to the number ofvariants of said ns-LTPs allergen and that in which said allergen is aParietaria Judaica allergen, specifically Parj1 (in any one formthereof) or ParJ2 (in any one form thereof).

In particular such compositions can be contacted with immunoglobulins by‘skin prick test’ in vivo, or on patient's tissues like, e.g., blood, invitro. Other modes of employ of the kit of the present invention todiagnostic ends are derivable by those skilled in the art in light ofthe knowledge of the state of the art. The invention will be betterdescribed with the aid of the attached figures.

DESCRIPTION OF THE FIGURES

FIG. 1 reports the amino acid sequences of the native Par J 1.0102 andPar j 2.0101 aligned therebetween and with respect to thethree-dimensional structure thereof. The notation of the amino acidsrelates to the sequence of the Par j 1.0102.

FIG. 2 shows the amino acid sequences of the Par j 1.0101 and of somens-LTP proteins aligned thereamong. The arrows indicate the disulfidebridges present in the three-dimensional structure of the proteins. Theamino acids are indicated in one-letter code. The Cs reported in thelast row of the table indicate the cysteine residues conserved in allproteins of the ns-LTP family.

FIG. 3 reports the schematic representation of the mutants of the majorallergen of the Parietaria Judaica Par j 1.0102, the sequence thereofbeing reported in the sequence listing as SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8 and SEQ ID NO: 10. The amino acids are reported inone-letter code. The underlined amino acids indicate the mutationseffected in the native sequence. The arrows indicate the disulfidebridges.

FIG. 4 reports in panel A the Western blot analysis results showing theIgE binding activity of the rParj1 and its disulfide bond variants byusing a pool of sera (n=30) from monosensitive Pj allergic patients.

Panel B shows a Coomassie Brilliant Blue staining of the recombinantproteins used.

In both panels on the first lane the result referred to the native Par j1.0101 is reported; on the second lane the result referred to the mutantPjA is reported; on the third lane the result referred to the mutant PjBis reported; on the fourth lane the result referred to the mutant PjC isreported; and on the fifth lane the result referred to the mutant PjD isreported.

FIG. 5 shows in panel A the outcome of a Western Blot analysis on a poolof sera from PJ allergic patients, aimed at demonstrating the IgEbinding capability (activity) of some mutants of the present inventionextensively disclosed in example 3.

On the first lane the result referred to the native Par j 1.0101 isreported; on the second lane the result referred to the mutant PjA isreported; on the third lane the result referred to the mutant PjB isreported; on the fourth lane the result referred to the mutant PjC isreported; and on the fifth lane the result referred to the mutant PjD isreported.

In panel B the outcome of a Western blot analysis on a pool of sera fromPJ allergic patients, aimed at demonstrating the IgG4 binding capabilityof the same abovedescribed mutants, it also extensively described inexample 3, is shown.

On the first lane the result referred to the native Par j 1.0101 isreported; on the second lane the result referred to the mutant PjA isreported; on the third lane the result referred to the mutant PjB isreported; on the fourth lane the result referred to the mutant PjC isreported; and on the fifth lane the result referred to the mutant PjD isreported.

FIG. 6 reports an histogram showing the results of the ELISA detectionexperiment carried out using the pool of sera used in example 3,extensively described on the example 4. Black histogram indicate resultsobtained with allergic sera; dotted square indicated results obtainedwith non allergic serum. On the y axis the optical density measured andin the x axis the proteins tested (the rParj1 and its disulfide bondvariants PjA, PjB, PjC, and PjD)) are reported.

FIG. 7 shows ten diagram reporting the results of the ELISA detectioncarried out using monosensitive sera from ten Pj allergic patientsextensively described on example 4, each diagram corresponding to arespective patient.

On each diagram on the y axis the optical density measured and in the xaxis the proteins used (the rParjl and its disulfide bond variants PjA,PjB, PjC, and PjD)) are reported.

On each diagram black squares indicate allergic sera, white squares anon allergic serum.

FIG. 8 reports an histogram showing the results of the ELISA detectionof the Ig binding activity of a rabbit polyclonal immune and pre-immuneantisera against rParj1, extensively described on the example 6.

On the y axis the optical density measured and in the x axis theantigens used (the rParj1 and its disulfide bond variants PjA, PjB, PjC,and PjD)) are reported.

Black squares indicate results obtained on rabbit immune, checkeredsquares results obtained on rabbit preimmune.

DETAILED DESCRIPTION OF THE INVENTION

The experimental approach that led to the present invention consisted ofa mutagenesis strategy aimed at the targeted disruption of all thedisulfide bridges present in all the ns-LTPs. This in order to generatemolecules with reduced or absent affinity to IgE, yet with intactaffinity to the other classes of antibodies apt to compete with thespecific IgEs against the native allergen. The ns-LTPs allergen ParJ1,whose sequence and structure is reported in comparison with Par J2 inFIG. 1, and in particular the isoform Par j 1.0102 (the primary sequencethereof being reported in the annexed sequence listing as SEQ ID NOs:1and 2), was taken as a molecular model, to be used for carrying outmutagenesis and the subsequent verification of the properties of theobtained mutant. In particular, recombinant DNA technology was resortedto in a strategy of site-directed mutagenesis against cysteines 4, 29,30, 50 and 52, i.e., the cysteines constituting the disulfide bridgesaccording to the structural model known to the art.

The results of these experiments demonstrate the close relationshipexisting between the three-dimensional structure of the protein and thecapability of forming epitopes for the IgEs, and in particular that thegradual disruption of the disulfide bridges causes a reduction of theserum IgEs binding activity thereof, whereas it does not affect the IgG4binding activity thereof.

This in light of the Western blot analysis described in example 3, andin FIG. 5, in which lanes 2, 3 and 4 (see FIG. 5 panel A) show thereduction of the serum IgEs binding activity by the mutants of thepresent invention, which should be construed as three-dimensionalmutants.

In particular, the Cys29-Cys30 mutant (PjA) shows a very weak bindingband (FIG. 5 lane 2) whereas the Cys50-Cys52 mutant (PjB) is somehowstill capable of binding the IgEs (FIG. 5, lane 3). Instead, moreremarkable is the result shown by the PjC and PjD mutants (FIG. 5, lanes4 and 5) for which no binding to human IgEs can be highlighted.

Such results have been confirmed by ELISA and IgE inhibition assays (seeexamples 4 and 5) where the PjB was the only variant still able to bindParj1-specific IgE antibodies in solution while the other variantsexhibited a very low inhibition capacity. The loss of additionaldisulfide bridges (PjC and PjD) leads- to the absence of any IgErecognition (see Example 4 and FIG. 4).

These results all together show that ns-LTP Parj1 variants lacking of atleast one disulfide bridge have a reduced allergenicity, which is evenabsent in the variants wherein the lacking bridge is localized in theaminoterminal region of the allergen at the domainalpha-helix1-loop1-alphahelix2, in particular when the lacking bridgesare at least two.

The maintenance by these variants of an overall antigenicity that,notwithstanding the reduced or absent allergenicity, is comparable oridentical to the one of the wild type allergen, has been shown byexperiments wherein binding activity of wild-type allergen and itsvariants to antibodies different than IgE, have been compared.

Such experiments are Western Blots carried out using as antibodiesrabbit policlonal antibodies and IgG4 of Pj allergic patients,extensively described in examples 6 and 3 respectively.

In these experiments the hypoallergenic variants generated by geneticengineering presented a similar behavior compared to the wild type, witha low reduction of their binding activity towards the anti-rParj1 rabbitantibodies.

Accordingly, a variant that lacks of at least one disulfide bridgesstill contains several protein domains similar to the native moleculeand, although at different extent, is apt to induce the production ofIgG antibodies (see example 3 and 6). IgE production and/or IgE-mediatedpresentation of the allergen, would be prevented by such “blocking”antibodies and reducing T cell proliferation and release of cytokines(25).

The above data have been confirmed also in vivo in particular by SkinPrick Test (SPT) analysis as described in example 7, where the purerecombinant proteins were tested on ten PJ allergic patients (the sameanalyzed by ELISA in example 4 and FIG. 7).

With regard to the single mutants, PjA showed a very low IgE bindingactivity and only 3 out of 10 patients with cutaneous Type Ihypersensitivity and a reduced wheal area respect to that one induced bywild-type allergen. On the contrary, loss of the Cys50-cys91 andCys4-Cys52 bridges seems to have aminor effect since an IgE bindingactivity and a positive SPT are still present. The loss of additionaldisulfide bridges (PjC and PjD) leads to the absence of any cutaneousreaction (see example 7).

These results obtained analyzing individuals demonstrate the reliabilityof the above described data in vitro, and above all that while thedisruption of the disulfide bridges in the amino terminal region (in thespecific case Cys4-Cys52 and Cys50-Cys91) affect even if not markedlythe human IgE binding capability of this mutant, the disruption of allthe four bridges (in the specific case Cys4-Cys52, Cysl4-Cys29,Cys30-Cys75 and Cys50-Cys91) have a devastating effect on the IgErecognition, and therefore on the allergenic response.

Accordingly, concerning the development of therapeutically usefulhypoallergenic molecules, variants lacking of three or four bridges, asin the specific case PjC and PjD mutants, are considered as preferredembodiments. The above was demonstrated using a pool of sera as well asa cohort of individual patients, indicating that the obtained result isrepresentative of the immune response of the allergic population.

It is pointed out that although obtained using specific variants derivedby mutating the wild type allergen, these results are in fact notlimited to the said specific variants, neither to the techniques usedfor the relevant derivation.

As such results are consequent to the modification of thethree-dimensional structure of the allergen, they could anyhow have beenobtained by any mutagenesis allowing the disruption of the disulfidebridges.

Accordingly any variant obtainable by deletion, substitution and/or theinsertion of one or more amino acidic residue which results in variantslacking of at least one disulfide bridge is included in the object ofthe invention.

The strategy of point mutation has however the remarkable advantage ofallowing the insertion of minimal variations at the level of the primarysequence of the native protein and therefore of generating mutants thatare more likely not to interfere with the variant recognition operatedby the T cells, and above all the possibility to generate proteinshaving a high reproducibility.

Variants having substantially the same length of the wild type areaccordingly considered preferred.

With regard to the techniques used for deriving the variants of theinvention, it is not limited to the genetic engineering ones, as theyare obtainable by techniques like chemical mutagenesis (formaldhehydeand gluteraldhehyde) which allow the disruption of disulfide bridgeseven in absence of mutations.

The genetic mutagenesis imply however the remarkable advantage ofallowing the generation of proteins having a high reproducibility, whilethe, chemical mutants do not ensure a denaturation pattern constant atevery preparation.

Furthermore, as the strategy described herein is independent on theepitope sequence on itself since it is based on the modification of thethree-dimensional structure of the IgE determinants, the adoptedmutagenesis strategy is actually independent from the primary sequenceof the allergen (and therefore from the sequence of the specific IgEepitopes).

For this reason variants of all the proteins with allergenic activitybelonging to the ns-LTP family (including Parj2), are included in theobject of the invention due to the conserved structure (cfr. e. g.s FIG.2 wherein Par j1 sequence is reported in comparison with the sequence ofother ns-LTPs together with the placement of the disulfides bridges).

In particular variant of the invention is not only any other mutein ofthe Parj1 allergen or of other ns-LTP allergens which, independentlyfrom the mutation carried out (substitution and/or deletion of one ormore amino acid residues) and of the way in which such a mutation iscarried out (e.g., by the above mentioned techniques), retain astructure equivalent to that of the corresponding native allergenlacking at least one disulfide bridge.

Thus the disruption of the disulfide bridges in ns-LTP allergensunderlies per se a limited or absent IgE binding ability of patientsallergic to the related variants.

Moreover, in particular in light to what is known in the art concerningthe high conservation of the structure and the cross-reactivity thathave led to the singling out of the so-called ns-LTP pan-allergen (seeabove) these data are indicative not merely of a suitability in thetherapy and prevention of the allergic forms caused by the allergenscorresponding to the individual variants, but also in the therapy andprevention of allergic forms caused by ns-LTP allergens other than thosecorresponding to the variants used.

A person skilled in the art can derive on the basis of his knowledge anyinformation suitable for deriving uses, compositions and kit describedin the summary of the invention.

With the aid of the following examples, a more detailed description ofspecific embodiments will now be given, in order to give a betterunderstanding of the objects, characteristics, advantages and operatingmethods of the present invention.

EXAMPLES Example 1 Cloning and Expression of Par J 1.0102

For the production of the major allergen of Parietaria Judaica Par j1.0102 the pQE30 prokaryotic vector (Qiagen) was used. The lattercharacteristically expresses recombinant proteins fused to a shorthistidine tail and inducible with isopropyl-p-D-thiogalactoside (IPTG).The histidine residues allow the purification of the recombinant proteinby affinity chromatography.

For this reason, 1 ng of the P5 clone containing the processed versionof the Par j 1.0102 (EMBL accession number X77414), the sequence thereofbeing reported in the annexed sequence listing as SEQ ID NO: 12, wassubjected to 30 cycles of polymerase chain reaction (PCR) amplificationat the following design: 94° C. for 1 min, 52° C. for 1 min, 72° C. for1 min. The synthetic primer oligonucleotides P5 forward and PS reverse,the sequence thereof being reported in the annexed sequence listing asSEQ ID NO:11 and SEQ ID NO: 12, respectively, were used.

The fragment thus generated was fractionated on 1% agarose gel in I×TBE,extracted, purified and digested with Bam H1 and Hind III restrictionenzymes and cloned in the Pqe30 VECTOR (Quiagen) previously digestedwith the same enzyme. The linearized vector and the digested fragmentswere incubated for 4 hours at 16° C. in presence of the enzyme DNAligase according to different stoichiometric ratios. The reactionmixture was then transformed in the bacterial strain MIS. Therecombinant clones were sequenced with the method of Sanger and thenucleotide sequence thus determined demonstrated that the DNA fragmentinserted into the pQE30 vector was identical to that known in the art(10).

Example 2 Cloning and Expression of Conformational Mutants of ParJ1.0102

PjA mutant (Cys29→Ser and Cys30→Ser) was generated using the TransformerSite-Directed Mutagenesis kit (Clontech) following the manufacturer'sinstruction and using the oligonucleotide PS (29,30) reported in thesequence listing as SEQ ID NO: 13 (mapping from nucleotide 88 tonucleotide 105) and the Parj1 sequence as a template. PjB mutant(Cys50→Ser and Cys52→Ser) was generated by PCR using as primers theoligonucleotide P5(50-52) reported in the sequence listing as SEQ ID NO:14 (mapping from nucleotide 91 to nucleotide 165) and PS reverseoligonucleotide and 1 ng of the Parj1 clone as a template. The PCRfragment was digested with Pst I and Hind III restriction enzymes andligated with the Pst I-Hind III linearized plasmid vector containing theParj1 sequence (expressing the first 31 amino acids of the wild type Parj 1.0102 allergen). PjC mutant (Cys4→Ser, Cys29→Ser and Cys30→Ser) wasgenerated by PCR amplification using the PjA variant as a template. Thecysteine residue at position 4 was mutated by PCR using theoligonucleotides P5(triple), the sequence thereof being reported as SEQID NO: 15, and P5 reverse.

After purification, PCR fragment was digested with Bam HI and Hind IIIenzymes and cloned in the pQE30 vector previously digested with the samerestriction enzymes. PjD mutant (Cys29→Ser, Cys30→Ser, Cys50→Ser andCys52→Ser) was generated using the Transformer Site-Directed Mutagenesiskit (Clontech) following the manufacturer's instruction and using thesynthetic oligonucleotide P5 (29,30) reported in the sequence listing asSEQ ID NO: 13 and the PjB variant as a template. All clones weresequenced with the method of Sanger (24) and the mutations and the openreading frames confirmed (See FIG. 3 for details).

With this process 4 independent mutants, hereinafter designated PjA (SEQID NO: 3 and SEQ ID NO: 4); PjB (SEQ ID NO: 5 and SEQ ID NO: 6), PjC(SEQ ID NO: 7 and SEQ ID NO: 8), and PjD (SEQ ID NO: 9 and SEQ ID NO:10) were isolated.

Example 3 Purification of Recombinant Proteins Evaluation of theRelevant Capability of Binding Ige of Allergic patients

10 ml O/N culture of the recombinant clones (NM15 strain, Quiagen) werethen used for an inoculation in 400 ml of 2YT broth (Bacto-tryptone 16gr/l, Bacto-yeast 10 gr/l, NaCl 5 gr/l, pH 7.0) containing ampicillinand kanamycin at a final concentration of 100 μgr/ml and 10 μgr/ml,respectively.

A 1:40 dilution was grown for 1 hour at 37° C. and, after that, inducedwith 1 mM isopropylthio-α-galactoside for 4 hours at 37° C. Cells wereharvested by centrifugation and the recombinant proteins purified byusing the His Trap kit (Pharmacia) following the manufacturer'sinstructions. Recombinant proteins, binding the HiTrap chelating column,were eluted using a buffer containing: 20 mM phosphate buffer pH7.4. 0.5M NaCl, 8 M UREA and 500 mM imidazole; fractions were analysed by 16%SDS-PAGE and Coomassie Brilliant Blue staining. Fractions containing thepurified protein were then diluted 1:100 in a buffer containing 20 mMphosphate buffer pH7.4. 0.5 M NaCl and 20 mM imidazole to allowrefolding of the protein, reloaded on the His Trap column and elutedwith a buffer with no denaturing agents (20 mM phosphate buffer pH7.4.0.5 M NaCl and 500 mM imidazole). Recombinant proteins were thendesalted using a centrifugal filter device (Centriprep, Millipore) andanalysed for their capability of binding human IgE from Pj allergicpatients by Western blot as previously described (12), using a pool ofsera (n=30) of Pj allergic patients which did not receive any specificimmunotherapy.

This analysis showed that the PjB mutant was still capable of bindinghuman IgE while the PjA mutant retains only a weak IgE binding activity.The PjC and PjD mutants did not show any IgE binding activity suggestingthat the IgE recognition was dependent on the three-dimensional foldingof the protein (FIG. 4 Panel A).

After that, membranes were stripped and reprobed with a His-tag specificreagent (INDIA™ Hisprobe-HRP, Pierce, USA) to check that theIgE-allergen complex was specific for the recombinant fused proteins.The concentration of the recombinant proteins was determined bydensitometric analysis of SDS-PAGE gels stained with Coomassie BrilliantBlue (see FIG. 4 panel B).

As a confirmation of this experiment another Western blot carried outusing IgE and IgG4 of allergic patients.

Then, the proteins purified were fractionated on 16% PAGE-SDS andtransferred on nitro-cellulose thanks to a Dry-blot system (Millipore).The membrane was incubated for 12-14 hours with a pool of sera from Pjallergic patients (1:5 dilution) in PBS-tween. The protein-human IgE andIgG4 binding complexes are highlighted using respectively a secondaryanti-IgE and anti-IgG4 antibodies conjugated to radish peroxidase. Thus,the complexes are highlighted using a chemioluminescence system(Super-signal, Pierce). The relevant results are reported in FIG. 4.

Example 4 Elisa Detection

The same pool of allergic sera from non-sensitive PJ allergic patientsused in example 3 has been used in an ELISA experiment, showing the IgEbinding activity of the rParj1 and its disulfide bond variants. A nonallergic subject has been tested as a negative control on the ELISA.

The results confirm the pattern of reaction of the experiment of theexample 3 (FIG. 4 Panel A) with the PjB variant reacting in a waycomparable to the wild-type allergen. A non allergic serum is shown as anegative control (FIG. 6).

The IgE binding activity of the four Parj1 disulfide bond variants wasalso tested by ELISA using sera from ten monosensitive Pj allergicpatients. Analysis of single sera showed a remarkable homogeneity of thereaction. In particular, the Cys4-Cys52 and Cys50-Cys91 bridges did notinfluence the allergenicity of the protein since this mutant (PjB)showed an IgE binding activity comparable to the wild-type allergen. Onthe other hand, the Cys14-Cys29 and Cys30-Cys75 bridges seem to becrucial for the IgE recognition. All the variants lacking those twobonds (PjA, PjC and PjD) presented low or even absent IgE bindingactivity. (FIG. 7)

ELISA detection has been performed by adding 200 μl of a solutioncontaining 5 μg/ml of antigen in coating buffer (sodium carbonate bufferpH 9.5) to each well of polystyrene plates overnight at roomtemperature. After several washing steps (1×PBS, 0.1% Tween 20) plateswere saturated with a solution containing 5% BSA, 0.5% Tween 20 incoating buffer. After washing, 200 μl of serum (1:5 dilution) from Pjallergic patients or from a non allergic subject were incubated for 4hours at room temperature. Bound IgE antibodies were detected with agoat antihuman IgE-HRP conjugate (Biosource International) diluted at aconcentration of 0.5 ng/ml in 1×PBS, 0.25% BSA, 0.1% Tween 20 for 1 hourat room temperature. After several washes, calorimetric reaction wasdeveloped by adding 0.2 ml/well of substrate solution (0.4 mg/mlo-phenylendiamine in 0.1 M citrate buffer).

Optical density was read at 495 nm in a BIO-RAD microplate reader.

Example 5 IgE Inhibition Assay

In order to investigate whether the disulfide bond variants were able toinhibit the binding of the IgE to the rparjl, increasing amount of eachrecombinant mutant were incubated with a pool of sera (n=10) of Pjmonosensitive allergic patients.

The ability of the Parj1 disulfide variants to interact with IgEantibodies was determined by an ELISA inhibition experiment. A pool ofsera (1:5 dilution) from ten monosensitive Pj allergic patients waspreincubated overnight with increasing concentration of each disulfidebond variant (0.25-20 μg/ml of protein). The solutions were added to theELISA wells coated with 5 mg/ml of rParj1 and the ELISA steps wereperformed as above described. Percentage of inhibition was calculatedaccording to the formula: %=100−OD_(A)/OD_(B)×100, where OD_(A) andOD_(B) represent the optical density read with the inhibited andnon-inhibited pool of sera respectively.

The results are reported in the following Table I TABLE I Inhibition ofIgE binding Protein tested % inhibition rParj1 95% PjA 16% PjB 85% PjC14% PjD 15%

The data reported in Table I suggests that all the variants lacking, atleast, Cys14-Cys29 and Cys30-Cys75 disulfide bonds exhibit a comparablelow level of inhibition (about 15%). On the contrary, the PjB variant(Cys50→Ser and Cys52ΔSer) showed a high percentage of inhibitionretaining a substantial ability of binding human IgE (about 85%).

Example 6 Rabbit Policlonal Binding Activity

Rabbits were immunized by PRIMM srl (Milan, Italy) using the rParj1allergen. As a control, rabbit polyclonal antibodies were analysed on aWestern blot using a Parietaria judaica crude extract detecting a bandof about 14000 Da corresponding to Parj1 native molecular weight. ELISAplates were coated at the same conditions as above described, with thewild-type Parj1 and with equal amount of each recombinant disulfide bondvariant, were probed with an anti-rParj1 specific polyclonal serum toanalyse their binding activity.

Rabbit preimmune and immune sera were diluted at a concentration of 6ng/ml and 200 μl of these solutions were incubated at room temperaturefor 1 hour. Wells were washed three times in 1×PBS, 0.1% Tween 20 andbound antibodies were detected using a donkey antirabbit Ig HRP linked(Amersham) at a 1:1000 dilution. Colorimetric reaction and opticaldensity were performed as above described.

The data obtained suggest that the PjA, PjB and PjC variants show asimilar behavior exhibiting a slight reduction of their binding ability(about 10%) compared to the Parj1 binding. The PjD variant showed areduced binding activity (about 20%) while the preimmune serum did notshow any reactivity towards the proteins (FIG. 8).

Example 7 Skin Prick Test Experiments with Purified Muteins

Ten patients, with a clear history of Parietaria judaica allergy andwith skin prick test (SPT) monosensitivity to Pj commercial extract,were analysed in this study. All the patients did not receiveimmunotherapy against Pj pollen and were not receivingglucocorticosteroid treatment. Allergens were used at 1 μg/mlconcentration diluted in 0.9% NaCl. About 20 μl of the test solution wasplaced on the forearms at a distance of more than 2.5 cm between eachprick. All tests were performed in duplicate. Histamine was used aspositive control and 0.9% NaCl solution as a negative control. Reactionswere measured after 20 min. By comparison with the wheal area generatedby histamine (100%), positive SPT were divided in three classes: 4+ wereassigned to SPT with an area >100% of area induced by histamine; 3+wereassigned to an area ≧80-100% and 2+ to an area ≧50-80%. Two non-allergicpatients (P.C. and D.G.) were tested as negative controls. Each subjectwas informed by the investigators and signed informed consent before thetest.

All patients showed a positive cutaneous reaction to the rParj1allergen. PjB was capable of inducing Type I immediate hypersensitivityin 9 out of 10 of the tested patients. PjA gave positive reaction in 3out of 10 of the patients and the wheal areas induced by prick werereduced in size respect to that ones triggered by the wild-typeallergen. The PjC and PjD did not give any SPT reaction. None reactionshave been observed when non allergic subjects were tested as reported inthe following table II. TABLE II Skin prick test of the rPar J and itsdisulfide bond variants Patient No. RParj1 PjA PjB PjC PjD  1 ++++ − − −−  2 +++ ++ +++ − −  3 ++++ − +++ − −  4 ++++ ++ +++ − −  6 ++++ − +++ −−  7 ++++ − ++++ − −  8 ++++ ++ +++ − −  9 +++ − ++ − − 10 +++ − ++ − −P.C. − − − − − D.G. − − − − −

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1. A variant of an allergen belonging to the family of the ns-LTPproteins, said variant maintaining most of the amino acid sequence ofthe wild type allergen, said variant lacking at least one of the fourdisulfide bridges constituting the structure of said allergen andexhibiting reduced IgE binding capability while maintaining thecapability of inducing IgG response.
 2. The variant according to claim1, said variant lacking at least one disulfide bridge in theamino-terminal region of said allergen.
 3. The variant according toclaim 1, said variant lacking two of said disulfide bridges constitutingthe structure of said allergen.
 4. The variant according to claim 1,said variant lacking three, or four of said disulfide bridgesconstituting the structure of said allergen.
 5. The variant according toclaim 1, said variant being a mutein wherein at least one of thecysteines constituting said disulfide bridges is deleted.
 6. The variantaccording to claim 1, said variant being a mutein wherein at least oneof the cysteines constituting said disulfide bridges is substituted withan amino acid residue not capable of forming disulfide bridges.
 7. Thevariant according to claim 6, wherein said amino acid residue is serineor alanine.
 8. The variant according claim 1, wherein said allergen isone of the major allergens of Parietaria Judaica.
 9. The variantaccording to claim 8, wherein said allergen is Parj 1, and saidcysteines constituting the disulfide bridges are the cysteines 4, 14,29, 30, 50, 52, 75 and
 91. 10. The variant according to claim 9, whereinthe sequence of said variant comprises a sequence selected from SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:
 7. 11. The variantaccording to claim 1 for use as a medicament or as a diagnostic agent.12. The variant according to claim 11 for use as a medicament in apatient customised treatment and/or prevention of allergic formassociated with an allergen belonging to the family of the ns-LTPproteins or as diagnostic agent for patient customised diagnosis ofallergic forms.
 13. The variant according to claim 12 which is a variantof a major allergen of Parietaria Judaica and wherein the allergic formis associated with an allergen of Parietaria Judaica.
 14. The variantaccording to claim 12, which has sequence selected from SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:
 7. 15. The variant accordingto claim 12 for use in the treatment of itch, erythema, edema, wheal,rash (urticaria) formation, rhino-conjunctivitis (seasonal allergies),bronchoconstriction, asthma and anaphylaxis.
 16. The variant accordingto claim 11 for use in preventive specific immunotherapy.
 17. Apolynucleotide coding for the variant according to claim
 1. 18. Thepolynucleotide according to claim 17, wherein the sequence of saidvariant comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6 and SEQ ID NO:
 8. 19. A vector comprising at least onepolynucleotide coding for the variant according to claim
 1. 20. A vectoraccording to claim 19, wherein the sequence of said variant comprises asequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQID NO:
 8. 21. A pharmaceutical composition comprising at least onevariant according to claim 1 and a pharmaceutically acceptable carrier.22. A pharmaceutical composition according to claim 21 wherein thevariant comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5 and SEQ ID NO:
 7. 23. A pharmaceutical compositioncomprising at least a polynucleotide, or a vector containing the same,coding for the variant according to claim 1 and a pharmaceuticallyacceptable carrier.
 24. A pharmaceutical composition according to claim23 wherein the sequence of said variant comprises a sequence selectedfrom SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO:
 8. 25. Adiagnostic agent comprising at least one variant according to claim 1and, optionally, a pharmaceutically acceptable carrier.
 26. A kit forthe derivation of a subject-customised allergogram for an allergic formassociated with an ns-LTP allergen, comprising a first compositioncomprising said ns-LTP allergen in native form together with anacceptable carrier; at least one composition comprising a single variantof said ns-LTP allergen according to claim 1 and an acceptable carrier;said allergogram being derivable contacting said compositions withimmunoglobulins of said subject and observing the effects thus obtained.27. The kit according to claim 26, comprising said first composition anda number of compositions each comprising a single variant of said ns-LTPallergen, equal to the number of the variants of said ns-LTP allergen.28. The kit according to claim 26, wherein said ns-LTP allergen isParJ1.